We will conduct a preliminary assessment of whether efficacy of erlotinib in NSCLC is reduced if tumors have high Notch pathway and CSC marker expression, and whether addition of the 3-secretase inhibitor RO4929097 will abrogate the negative impact of high Notch/CSC marker expression. We will also assess whether Notch and CSC markers correlate with EMT and EGFR markers, and whether baseline marker signatures predict for efficacy of erlotinib alone or the RO4929097-erlotinib combination. We will assess whether exposure to erlotinib alone enriches tumors for Notch/CSC/EMT markers as a mechanism of acquired resistance, and whether addition of RO4929097 to erlotinib prevents this enrichment or alters impact of erlotinib on EGFR pathway signaling. We will assess impact of host Notch SNPs on tumor Notch/ CSC/ EMT/ EGFR marker expression and on efficacy of erlotinibRO4929097. We may also assess them as a secondary goal. Specifically, where tissue quantity and patient numbers permit, we will conduct preliminary assessments of: a) Notch/CSC/EMT marker expression in tumors with vs without other erlotinib resistance factors (eg, T790M mutation, IGF-1R activation, c-Met amplification);b) how Sonic Hedgehog (SHH) and Wnt pathway components modulate or augment the impact of the Notch pathway on EGFR TKI efficacy;c) Notch/CSC/EMT marker expression in EGFR-mutant vs -wild type tumors. Specific Aim 1: We will obtain pre-treatment biopsies from patients receiving erlotinib RO4929097 and will assess Notch pathway, CSC, EMT and EGFR pathway markers by immunohistochemistry (IHC) and reverse phase protein arrays (RPPAs). We will: a) correlate expression of Notch and CSC markers with each other and with EMT and EGFR markers;b) correlate marker expression with tumor shrinkage after RO4929097 initiation;c) correlate marker expression with tumor shrinkage after erlotinib initiation;d) compare marker expression in patients with vs without recent erlotinib exposure Specific Aim 2: We will obtain repeat NSCLC biopsies 6 weeks after initiating therapy with erlotinib RO4929097 and will compare change in Notch, CSC, EMT and EGFR marker expression in patients who received erlotinib alone to those who also received RO4929097. Specific Aim 3: We will assess how host Notch pathway SNPs (assessed in peripheral blood mononuclear cells [PBMCs]) correlate with: a) tumor expression of Notch, CSC, EMT and EGFR markers;b) change in tumor markers with erlotinib RO4929097;c) efficacy of erlotinib RO4929097. PUBLIC HEALTH RELEVANCE: Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases. About 10% of NSCLCs have activating mutations of the epidermal growth factor receptor (EGFR) gene, and tumors with these mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib. We are conducting an NCI-sponsored trial in which erlotinib-resistant NSCLC patients have the 3-secretase/ Notch inhibitor RO4929097 added to their erlotinib. While erlotinib is less effective vs EGFR wild type tumors, therapeutic benefit is nevertheless seen.